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. 2017 Apr 11;7:46208.
doi: 10.1038/srep46208.

Tomatidine Enhances Lifespan and Healthspan in C. Elegans Through Mitophagy Induction via the SKN-1/Nrf2 Pathway

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Free PMC article

Tomatidine Enhances Lifespan and Healthspan in C. Elegans Through Mitophagy Induction via the SKN-1/Nrf2 Pathway

Evandro F Fang et al. Sci Rep. .
Free PMC article

Abstract

Aging is a major international concern that brings formidable socioeconomic and healthcare challenges. Small molecules capable of improving the health of older individuals are being explored. Small molecules that enhance cellular stress resistance are a promising avenue to alleviate declines seen in human aging. Tomatidine, a natural compound abundant in unripe tomatoes, inhibits age-related skeletal muscle atrophy in mice. Here we show that tomatidine extends lifespan and healthspan in C. elegans, an animal model of aging which shares many major longevity pathways with mammals. Tomatidine improves many C. elegans behaviors related to healthspan and muscle health, including increased pharyngeal pumping, swimming movement, and reduced percentage of severely damaged muscle cells. Microarray, imaging, and behavioral analyses reveal that tomatidine maintains mitochondrial homeostasis by modulating mitochondrial biogenesis and PINK-1/DCT-1-dependent mitophagy. Mechanistically, tomatidine induces mitochondrial hormesis by mildly inducing ROS production, which in turn activates the SKN-1/Nrf2 pathway and possibly other cellular antioxidant response pathways, followed by increased mitophagy. This mechanism occurs in C. elegans, primary rat neurons, and human cells. Our data suggest that tomatidine may delay some physiological aspects of aging, and points to new approaches for pharmacological interventions for diseases of aging.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Tomatidine extends lifespan and healthspan in C. elegans.
(A) Lifespans of tomatidine treated C. elegans were assessed (n = 91 for vehicle and n = 126 for Tomatine group). (B,C) Healthspan metrics of pharyngeal pumping (B) and swimming movement (C) for C. elegans treated with 25 μM tomatidine (n = 10–20). (D,E) Morphology of C. elegans pharynx was assessed using machine learning technique to determine age state of pharynx (D, n = 30 worms in three separate experiments), with a representative set of pictures shown (E). (F) Metabolomics showing the relative change in C. elegans metabolome with or without tomatidine. Adult D7 worms from tomatidine or vehicle-treated groups were collected and subjected to ALEX-CIS GCTOF mass spectrometry with more than 150 metabolites identified. See Table S1 for a complete list. All bar graphs are expressed as mean ± S.E.M., *p < 0.05; **p < 0.01; ***p < 0.001. See also Figures S1 and S2.
Figure 2
Figure 2. Effects of tomatidine on the transcriptome of C. elegans.
(A) Heatmap of microarray data showing genes that were differentially expressed in control and tomatidine-treated nematodes. (B) A list of significantly changed GO signaling pathways in tomatidine-treated worms compared with vehicle-treated changes. Mitochondria-related pathways are marked orange. See Table S2 for a complete gene list.
Figure 3
Figure 3. Tomatidine modulates mitochondrial homeostasis by stimulating both mitochondrial biogenesis and mitophagy.
(A) Assessment of mitochondrial content in nematode pharynx determined by quantifying mitoTracker Red fluorescence (n = 15). Representative images are also shown. (B,C) Monitoring of mitochondrial network of the pharynx muscle cells in vehicle- and tomatidine-treated myo-3::GFP nematodes. Representative images are shown in panel B, and quantification of mitochondrial network scoring is shown in panel C (n = 40). (D) Immunoblot showing relative levels of the indicated mitochondrial proteins in control and tomatidine-treated human neural cells. For full-length gels see Figure S4A. (E) Detection of muscle cell mitophagy in vehicle- and tomatidine-treated nematodes. The mitophagy-screening strain N2;Ex(pmyo-3::dsred::lgg-1;pdct-1::dct-1::gfp) was used for mitophagy detection. A set of representative images is shown. (F,G) Assessment of mitophagy in vehicle- and tomatidine-treated primary rat cortical neurons. Colocalization between the mitophagy dye and the lysosome dye were quantified (F) with a representative set images shown in panel G. (H,I) Assessment of mitophagy in vehicle- and tomatidine-treated human neural cells. Colocalization between the mitophagy dye and the lysosome dye were quantified (H) and representative images are shown in panel I. (J,K) Evaluation of mitophagy in vehicle- and tomatidine-treated HeLa cells expressing mt-mKeima (see Supplementary Methods for details). Ratios indicating relative levels of mitophagy were quantified (J) and representative images are shown in panel K. (L) Evaluation of C. elegans basal OCR was performed using a Seahorse XF96 instrument (mean ± S.E.M with n = 10 replicates/group). See also Figure S2.
Figure 4
Figure 4. Mitophagy is necessary for tomatidine-induced stress resistance in worms.
(A) dct-1 knockout nematodes were treated with 25 μM of tomatidine and pharyngeal pumping was assessed at day 5 and day 9 (mean ± S.E.M., n = 39–40). (B) Day 4 nematodes treated with or without tomatidine were exposed to the mitochondrial toxin rotenone (25 μM) for 2 days and survival was quantified on day 6 (mean ± S.E.M, n = 41–61). (C) Day 4 nematodes treated with or without tomatidine were exposed to heat stress (37 °C for 7 h), and survival was quantified (mean ± S.E.M, n = 41–61).
Figure 5
Figure 5. Tomatidine-induced mitophagy is mediated by ROS and activation of Nrf2/SKN-1.
(A) Assessment of tomatidine-induced Nrf2/ARE activity in ARE-bla HEK-293 cells (mean ± S.E.M., n = 6). (B) Immunoblot showing effects of tomatidine on levels of the indicated proteins in human neural cells. Cells were treated with the indicated concentrations of tomatidine for 24 h. For Nrf2, the nuclear fraction was isolated and used for immunoblotting. For full-length gels see Figure S4B. (C) Changes of cellular ROS in WT and Nrf2 knockdown human neural cells treated with or without 4 μM tomatidine. Cells were stained with DHE (an indicator of superoxide levels) followed by FACS analysis (mean ± S.E.M, n = 6). (D) Assessment of mitophagy in WT and Nrf2 knockdown human neural cells treated with or without 4 μM tomatidine (24 h) or a ROS scavenger NAC (1 mM for 3 h) (mean ± S.E.M, n = 6). (E) Effects of tomatidine on pharyngeal pumping rate in N2 wild type and skn-1 (zu135) C. elegans (mean ± S.E.M, n = 30). See also Figure S3. (F) Pharyngeal pumping rate in both N2 wild type and atfs-1 RNAi knockout C. elegans (mean ± S.E.M, n = 20).

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